Deoxyribonuclease, gene encoding same and use thereof

ABSTRACT

This invention provides a novel acid DNase (DLAD) which is an endonuclease capable of cleaving DNA independently from divalent cations, under acidic conditions, which retains its activity in acidic to even neutral pH range, and which is not inhibited by G-actin. This invention also provides a DNA encoding the enzyme, an expression vector containing the DNA, and a host cell transformed with the expression vector. Furthermore, a pharmaceutical composition containing DLAD, DLAD expression vector or a host cell transformed with the expression vector as an active ingredient is provided. The pharmaceutical composition is useful as a therapeutic agent replacing DNase I for cystic fibrosis, and can provide a new approach for the prophylaxis and treatment of infectious diseases.

TECHNICAL FIELD

This invention relates to a novel deoxyribonuclease capable of cleavingDNA independently from divalent cations under acidic conditions, a DNAencoding same and use of these for the prophylaxis and treatment ofinfectious diseases as well as for the treatment of cystic fibrosis.

BACKGROUND ART

The presence of various deoxyribonucleases (hereinafter referred to asDNase) in mammalian cells has been known. DNase II is one of the DNasesstudied most and catalyzes DNA hydrolysis reaction in the absence ofdivalent cations at acidic pH [in The Enzymes (Boyer, P. D., ed) 3rdEd., Vol. 4, pp. 271–287 (1971), Academic Press, New York; Arch.Biochem. Biophys., 300: 440–450 (1993)]. While the acid DNase activitiesare widely found in various animal tissues [Biochim. Biophys. Acta,1119: 185–193 (1992); J. Biol. Chem., 273: 2610–2616 (1998)], DNase IIhas been considered to be the sole enzyme responsible for the acid DNaseactivities. Because DNase II shows low organ specificity and isdistributed ubiquitously, a possibility of DNase II playing an importantbiological role in the fundamental biological phenomena, such as DNAcatabolism and apoptosis, has been suggested [The Enzymes (1971), supra;Arch. Biochem. Biophys., 300: 440–450 (1993)].

Even though the enzymological properties of the DNase II isolated fromdifferent organisms are very similar, their physicochemical propertiesand molecular structures are strikingly different. For example, it isknown that porcine DNase II is a complex protein consisting ofunidentical subunits derived from its precursor protein, but DNase IIderived from other animals are mostly single polypeptides [J. Biol.Chem., 260: 10708–10713 (1985); Biochem. Biophys. Res. Commun., 247:864–869 (1998); J. Biol. Chem., 251: 116–123 (1976); Gene, 215: 281–289(1998)]. Furthermore, the apparent molecular weights of DNase II varyfrom 26.5 kDa to 45 kDa [J. Biol. Chem. (1976), supra; Gene, (1998),supra; J. Biol. Chem., 247: 1424–1432 (1972); Eur. J. Biochem., 202:479–484 (1991)].

The diversity of acid DNases can be also appreciated from thesubcellular localization. DNase II is considered to be localized inlysosomes [J. Biol. Chem. (1972), supra; Biochim. Biophys. Acta, 1007:15–22 (1989)], but acid DNase activity is also found in nuclear fraction[Arch. Biochem. Biophys. (1993), supra; Biochem. J., 136: 83–87 (1973)].

The reason for such molecular diversity of DNase II still remainsunclear, but the aforementioned findings suggest the existence of adifferent acid DNase distinguishable from DNase II. In fact, the presentinventors have identified and partially purified novel acid DNases(DNase α and DNase β) from the nuclear fraction of rat thymus (JP8-187079 A). In view of the foregoing situation it is considered to becritical for the elucidation of the diversity of acid DNases to searchother novel acid DNases and determine their characteristics.

In addition, DNase has been actively studied with the aim of applyingsame for the prophylaxis and treatment of various diseases. One of theclinical applications of DNase, which has been drawing particularattention in recent years, is an application to the treatment of cysticfibrosis (hereinafter sometimes to be also referred to as CF) [Annu.Rev. Pharmacol. Toxicol., 35: 257–276 (1995); Chest, 107: 65–70 (1995)].CF is a lethal hereditary disease caused by abnormal chloride ionchannel of exocrine glands. In the Caucasian population, one in 2500newborns suffers from this disease and one in 25 Caucasians is acarrier. About 90% of the CF patients die of respiratory insufficiencycaused by intractable infection with Pseudomonas aeruginosa in theinferior airway in their 20's and 30's [Curr. Opin. Pulm. Med., 6:425–434 (1995)]. Phlegm that is accumulated in the airway to impair therespiratory function is caused by high concentration DNA released fromthe disrupted leukocytes infiltrating into the inflammatory site.Genentech, Inc. U.S. is selling a recombinant DNase I as a therapeuticagent for CF in Europe and America, which aims at removing the highmolecular weight DNA accumulated in the lung, recovering the respiratoryfunction and preventing infectious diseases [Annu. Rev. Pharmacol.Toxicol. (1995), supra; Chest (1995), supra]. DNase I not only degradesDNA, but also depolymerizes F-actin which is abundant in the sputum ofCF patients. However, since the resulting monomeric G-actin stronglyinhibits DNase I, DNase I is immediately inactivated. Actually, DNase Ihardly shows any therapeutic effect. Some attempts have been made toproduce a G-actin nonsensitive DNase I by genetic recombination, butsatisfactory DNase has not been obtained yet [Proc. Natl. Acad. Sci.USA, 93: 8225–8229 (1996); J. Biol. Chem., 273: 18374–18381 (1998)].Thus, there is a demand on the identification of a novel G-actinnonsensitive DNase effective for the treatment of CF.

A second interest in the clinical application of DNase is that for theprophylaxis and treatment of infectious diseases. Some DNases areconsidered to play an important role in the biological defensemechanisms against infection with bacteria and viruses, based ondegradation of foreign genomic DNAs. Accordingly, identification of theDNase involved in the prevention of infection in mammals, such as human,and utilization thereof as a medicament are expected to open a newpossibility in the prophylaxis and treatment of infectious diseases.

It is therefore an object of the present invention to provide a novelacid DNase and clarify the characteristics of the enzyme, therebyproviding critical information for the study of the molecular diversityof acid DNases. It is another object of the present invention to providea novel G-actin nonsensitive DNase that can be effectively used as atherapeutic agent of CF. It is yet another object of the presentinvention to provide a novel DNase useful for the prophylaxis andtreatment of infectious diseases.

DISCLOSURE OF THE INVENTION

In an attempt to accomplish the above-mentioned objects, the presentinventors have conducted intensive studies, and succeeded in isolatingcDNA clones containing an ORF encoding a novel protein homologous tohuman DNase II, from RNA derived from the liver of human, mouse or rat.Furthermore, it has been confirmed that this protein has an endonucleaseactivity capable of cleaving the DNA independently from divalent cationsunder acidic conditions, like DNase II, but is a novel acid DNasedistinguishable from DNase II in the capability of exerting the DNaseactivity even in the neutral pH range and the sensitivity againstdivalent metallic ion inhibitors, as a result of the analysis of thephysicochemical and enzymological characteristics of the proteinobtained by culturing a host cell transformed with an expression vectorcontaining the cDNA clone and purifying the recombinant protein. Then,the present inventors have designated the novel acid DNase as DLAD(DNase II-Like Acid DNase). The present inventors have also demonstratedthat this enzyme has a high possibility of making an effectivetherapeutic agent of CF by confirming that the DLAD activity is notinhibited by G-actin. Moreover, the present inventors have confirmed ahigh possibility of the DLAD having a preventive effect on viralinfectious diseases, which resulted in the completion of the presentinvention.

Accordingly, the present invention provides the following.

-   (1) A DNase which is an endonuclease capable of cleaving DNA    independently from divalent cations under acidic conditions and    having the following properties:-   (1) active pH range: ca. 4.0 to ca. 7.6-   (2) DNA cleavage mode: 3′-P/5′-OH end forming type-   (3) sensitivity against inhibitors:-   (i) inhibited by Zn²⁺-   (ii) not inhibited by G-actin-   (2) The DNase of (1) above, further having the following properties:-   (1) optimal pH: ca. 5.2-   (2) molecular weight: ca. 55 kDa as a post-translational    modification product (SDS-PAGE)-   (3) localization: present in cytoplasm and extracellularly, rich in    cytoplasm-   (4) tissue specificity: specifically expressed in the liver.-   (3) A DNase having the following polypeptide (a) or (b):-   (a) a polypeptide consisting of an amino acid sequence of amino acid    Nos. 1 to 332 of the amino acid sequence shown in Sequence Listing,    SEQ ID NO: 1-   (b) a polypeptide having the same amino acid sequence of (a) above,    except that one to several amino acids are deleted, substituted,    inserted, added or modified, wherein a mature protein has an    endonuclease activity capable of cleaving a DNA independently from    divalent cations in a pH range of from ca. 4.0 to ca. 7.6.-   (4) The DNase of any of (1) to (3) above, wherein a primary    translation product contains an N terminal signal peptide sequence,    preferably an amino acid sequence of the amino acid Nos. −22 to −1    of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1.-   (5) The DNase of any of (1) to (4) above, which is derived from a    mammal, preferably mouse.-   (6) A DNase having the following polypeptide (a) or (b):-   (a) a polypeptide consisting of an amino acid sequence of the amino    acid Nos. 1 to 334 of the amino acid sequence shown in Sequence    Listing, SEQ ID NO: 3.-   (b) a polypeptide having the same amino acid sequence of (a) above,    except that one to several amino acids are deleted, substituted,    inserted, added or modified, wherein a mature protein has an    endonuclease activity capable of cleaving a DNA independently from    divalent cations in a pH range of from ca. 4.0 to ca. 7.6.-   (7) The DNase of any of (1), (2) and (6) above, wherein a primary    translation product contains an N terminal signal sequence,    preferably an amino acid sequence of the amino acid Nos. −27 to −1    of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3.-   (8) The DNase of (1), (2), (6) or (7) above, which is derived from a    mammal, preferably human.-   (9) A DNA encoding the DNase of any of (1) to (8) above.-   (10) A DNA consisting of the following nucleotide sequence (a) or    (b):-   (a) a nucleotide sequence of the nucleotide Nos. 279 to 1274 of the    nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2-   (b) a nucleotide sequence capable of being hybridized to the    nucleotide sequence of (a) above under stringent conditions, which    encodes a DNase having an endonuclease activity capable of cleaving    DNA independently from divalent cations in a pH range of from ca.    4.0 to ca. 7.6.-   (11) A DNA consisting of the following nucleotide sequence (a) or    (b):-   (a) a nucleotide sequence of the nucleotide Nos. 213 to 1274 of the    nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2-   (b) a nucleotide sequence capable of being hybridized to the    nucleotide sequence of (a) above under stringent conditions, which    encodes a primary translation product of a DNase whose mature    protein has an endonuclease activity capable of cleaving DNA    independently from divalent cations in a pH range of from ca. 4.0 to    ca. 7.6.-   (12) The DNA of (10) or (11) above, which is derived from a mammal,    preferably mouse.-   (13) A DNA consisting of the following nucleotide sequence (a) or    (b):-   (a) a nucleotide sequence of the nucleotide Nos. 82 to 1083 of the    nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4-   (b) a nucleotide sequence capable of being hybridized to the    nucleotide sequence of (a) above under stringent conditions, which    encodes a DNase having an endonuclease activity capable of cleaving    DNA independently from divalent cations in a pH range of from ca.    4.0 to ca. 7.6.-   (14) A DNA consisting of the following nucleotide sequence (a) or    (b):-   (a) a nucleotide sequence of the nucleotide Nos. 1 to 1083 of the    nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4.-   (b) a nucleotide sequence capable of being hybridized to the    nucleotide sequence of (a) above under stringent conditions, which    encodes a primary translation product of a DNase whose mature    protein has an endonuclease activity capable of cleaving DNA    independently from divalent cations in a pH range of from ca. 4.0 to    ca. 7.6.-   (15) The DNA of (13) or (14) above, which is derived from a mammal,    preferably human.-   (16) A recombinant vector containing the DNA of any of (9) to (15)    above.-   (17) An expression vector containing the DNA of any of (9) to (15)    above and a promoter operably linked to said DNA.-   (18) A transformant obtained by transforming a host cell with the    expression vector of (17) above.-   (19) A method for producing the DNase of any of (1) to (8) above,    which comprises culturing the transformant of (18) above in a medium    and recovering said DNase from the resulting culture.-   (20) A pharmaceutical composition containing the DNase of any of (1)    to (8) above, the expression vector of (17) above or the    transformant of (18) above as an active ingredient.-   (21) The pharmaceutical composition of (20) above, which is for the    prophylaxis and treatment of infectious diseases or for the    treatment of cystic fibrosis.

Inasmuch as the DLAD of the present invention is an acid DNase thatexpresses the activity in a broad pH range of from acidic to neutral pHsindependently from divalent cations, and is resistant to G-actin, it isuseful for degrading a high concentration DNA contained in the sputum ofCF patients, improving the respiratory function.

Furthermore, because the DLAD of the present invention can suppress theintracellular expression of foreign genes, it also provides a usefulmeans for the prophylaxis and treatment of infectious diseases, such asviral infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an intracellular/extracellular (medium) presence ratio (%of the total) of each protein in HeLa S3 cells engineered to expressDLAD, DNase II signal-DLAD chimeric protein or DNase II, wherein a blackline shows intracellular presence and a white line shows extracellularpresence.

FIG. 2 shows sensitivity of DLAD to various DNase inhibitors, wherein A(∘): MgCl₂, (●): MgSO₄; B: aurintricarboxylic acid; C: G-actin; D (▪):CoCl₂, (Δ): NiCl₂, (●): ZnCl₂.

FIG. 3 shows GFP or β-galactosidase activity in HeLa S3 cellsco-transfected with a DLAD or DNase II expression vector and a GFP(closed column) or β-galactosidase (open column) expression vector. Theactivities are shown as the ratio (%) to the activity in the HeLa S3cells co-transfected with a control vector and a reportor geneexpression vector. The data are the average values (column) of 3independent experiments ± standard error (error bar).

BEST MODE FOR CARRYING OUT THE INVENTION

The DLAD of the present invention is similar to DNase II, which is aknown acid DNase, in that it has an endonuclease activity thathydrolyzes DNA to generate 3′-P/5′-OH termini under acidic conditionsindependently from divalent cations. However, DLAD is extremelycharacteristic in that it exerts DNase activity over a wide pH range offrom pH ca. 4.0 to ca. 7.6, whereas DNase II shows activity only in a pHrange of not more than ca. 5.6. The pH range preferable for the DLADactivity is from ca. 4.4 to ca. 6.8, and the optimal pH is about 5.2.

DLAD is also characteristically different from DNase II in thesensitivity to divalent metal ions. To be specific, DLAD issignificantly sensitive to Zn²⁺ as compared with Co²⁺, Ni²⁺ and thelike, whereas Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ and the like influence DNase IIactivity to almost the same level.

The DLAD of the present invention is not inhibited by G-actin, unlikeDNase I, etc.

The DLAD of the present invention is not particularly limited as long asit has the above-mentioned characteristics, and the origin of DLAD isnot limited, either. Thus, it encompasses not only those originated fromnaturally occurring organisms but also spontaneous or artificial mutantsor those derived from transformants obtained by introducing aheterologous (i.e., foreign) DLAD gene. Preferably, it includes DLADderived from mammals, such as human, bovine, porcine, horse, monkey,sheep, goat, canine, feline, rabbit, mouse, rat, guinea pig and thelike. Those derived from human, bovine, porcine, mouse and rat areparticularly preferable.

The DLAD of the present invention can have various molecular weights bychanging the amino acid composition or by glycosylation, and preferablyhas a molecular weight of about 38 to 39 kDa (calculated) when it is anunglycosylated mature polypeptide chain, and about 55 kDa (SDS-PAGE)when it is a glycosylated mature protein (post-translationalmodification product). When DLAD is translated as a precursor containinga signal peptide sequence, it preferably has a molecular weight of about41 to 42 kDa when it is a primary translation product.

In an embodiment of the present invention, DLAD is distributed bothextracellularly and in cytoplasm. More specifically, DLAD is presentmainly in cytoplasm, and shows a presence ratio different from that ofDNase II. In this embodiment, as is expected from additionalextracellular secretion, the primary translation product of DLADcontains a signal peptide sequence at its N terminus. The signal peptideis not particularly limited as long as it is recognized and cleaved by asignal peptidase in endoplasmic reticulum, resulting in a mature DLADprotein. Examples thereof include an amino acid sequence of the aminoacid Nos. −22 to −1 of the amino acid sequence shown in SequenceListing, SEQ ID NO: 1, an amino acid sequence of the amino acid Nos. −27to −1 of the amino acid sequence shown in Sequence Listing, SEQ ID NO:3, and an amino acid sequence obtained by deleting, substituting,inserting or adding one to several amino acids of these amino acidsequences as long as the property of a signal sequence is generallyunderstood to be retained. Signal sequences of other secretory proteins,such as DNase II, are also preferable.

It is not particularly limited where in the substructure of cytoplasm acytoplasmic DLAD is localized, but it is preferably localized in one ofmore organelles in an acidic environment, such as lysosomes andperoxysomes.

The expression of the DLAD of the present invention is highly restrictedto that in the liver, making a sharp contrast with DNAse II which is lowin organ specificity.

In a preferable embodiment of the present invention, DLAD is apolypeptide consisting of an amino acid sequence of the amino acid Nos.1 to 332 of the amino acid sequence shown in Sequence Listing, SEQ IDNO: 1 or a polypeptide consisting of an amino acid sequence of the aminoacid Nos. 1 to 334 of the amino acid sequence shown in Sequence Listing,SEQ ID NO: 3, or a polypeptide which consists of the same amino acidsequence of these, except that one to several amino acids are deleted,substituted, inserted, added or modified, and which has an endonucleaseactivity capable of cleaving DNA in a pH range of from ca. 4.0 to ca.7.6 independently from divalent cations.

The DLAD of the present invention can be obtained by appropriatelyemploying (1) a method including extraction and purification from thecells or tissues, that produce this enzyme, as a starting material, (2)a method including chemical synthesis or (3) a method includingpurification from the cells engineered to express DLAD by geneticrecombination techniques, or the like.

For example, the isolation and purification of DLAD from a naturallyoccurring DLAD-producing tissue can be carried out as follows. Amammalian tissue (e.g., a liver tissue section from human, mouse, rat,etc.) is homogenized in a suitable extraction buffer, ultrasonicated ortreated with a surfactant to give a cell extract, and purified by asuitable combination of separation techniques conventionally utilizedfor separation and purification of proteins. Examples of the separationtechnique include methods utilizing difference in solubility, such assalting out and solvent precipitation, methods utilizing difference inmolecular weight, such as dialysis, ultrafiltration, gel filtration,non-denatured polyacrylamide gel electrophoresis (PAGE) and sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), methodsutilizing charge, such as ion exchange chromatography and hydroxyapatitechromatography, methods utilizing specific affinity, such as affinitychromatography, methods utilizing difference in hydrophobicity, such asreversed phase high performance liquid chromatography, methods utilizingdifference in isoelectric point, such as isoelectric focusing, and thelike.

Alternatively, DLAD can be obtained by culturing mammal-derived culturedcells, for example, cultured cells derived from liver cells of human,mouse, rat and the like, in a suitable liquid medium and purifying fromthe obtained culture supernatant by the above-mentioned conventionalprotein separation techniques.

The production of DLAD by chemical synthesis can be carried out by, forexample, synthesizing the whole or partial sequence based on an aminoacid sequence (amino acid Nos. 1 to 332) shown in Sequence Listing, SEQID NO: 1 or an amino acid sequence (amino acid Nos. 1 to 334) shown inSequence Listing, SEQ ID NO: 3 using a peptide synthesizer andrenaturing the obtained polypeptide under suitable renaturationconditions.

The DLAD of the present invention is preferably produced by cloning aDNA encoding the protein, and isolating and purifying from the cultureof a transformant containing an expression vector carrying the DNA.

The cloning of an enzyme gene is typically performed as follows. First,a desired enzyme is completely or partially purified from cells ortissues producing said enzyme using the above-mentioned means, followedby Edman method to determine its N terminal amino acid sequence.Furthermore, the enzyme is partially degraded with proteases or chemicalsubstances that cleave a peptide in a sequence specific manner, and theamino acid sequence of the obtained oligopeptide is determined in thesame manner by Edman method. The oligonucleotides having the nucleotidesequences corresponding to the determined amino acid sequences aresynthesized, and using them as probes, a DNA encoding said enzyme iscloned from a cDNA or genomic DNA library prepared from the cells ortissues that produce said enzyme by colony (or plaque) hybridizationmethod.

Alternatively, an antibody against the enzyme is produced according to aconventional method using, as an antigen, the entirety or a part of thecompletely of partially purified enzyme, and a DNA encoding said enzymecan be cloned by antibody screening method from a cDNA or genomic DNAlibrary prepared from the cells or tissues, that produce this enzyme.

When a gene encoding an enzyme, whose enzymological properties aresimilar to those of the enzyme of interest, is known, a DNA encodingsaid enzyme can be cloned by searching EST (Expressed Sequence Tag)clones of mammals, such as human, mouse and rat, registered on generallyavailable databases, such as EMBL and GenBank, extracting a clone thatshows homology to the nucleotide sequence of the known gene, producingprobes as mentioned above, based on the nucleotide sequence of theextracted EST clone, and carrying out colony (or plaque) hybridization.In the case of the DLAD of the present invention, an EST clone, which isa fragment of cDNA encoding DLAD, can be found by a homology searchusing a nucleotide sequence encoding DNase II derived from mammals suchas human.

Alternatively, RACE method can be used to obtain a cDNA clone morerapidly and easily. To be specific, an EST clone corresponding to a partof the DLAD gene is extracted as mentioned above, oligonucleotideshomologous to the partial nucleotide sequences of sense and antisensestrands of said EST clone are respectively synthesized. Using eacholigonucleotide and an appropriate adaptor primer as a pair of PCRprimers, 5′ and 3′ RACE reactions are carried out, and eachamplification fragment is ligated by a method using a restriction enzymeand a ligase to give a full length cDNA clone.

The nucleotide sequence of the DNA obtained as mentioned above can bedetermined using known sequencing techniques such as Maxam-Gilbertmethod and dideoxy termination method.

The DNA encoding DLAD of the present invention is preferably a DNAencoding an amino acid sequence of the amino acid Nos. 1 to 332 of theamino acid sequence shown in Sequence Listing, SEQ ID NO: 1 or an aminoacid sequence of the amino acid Nos. 1 to 334 of the amino acid sequenceshown in Sequence Listing, SEQ ID NO: 3, or the same amino acidsequences of these, except that one to several amino acids are deleted,substituted, inserted, added or modified, wherein a protein consistingof said mutated amino acid sequence has an endonuclease activity capableof cleaving DNA in a pH range of from ca. 4.0 to ca. 7.6 independentlyfrom divalent cations. More preferably, the DNA encoding DLAD of thepresent invention is one which consists of a nucleotide sequence of thenucleotide Nos. 279 to 1274 of the nucleotide sequence shown in SequenceListing, SEQ ID NO: 2 or a nucleotide sequence of the nucleotide Nos. 82to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO:4, or a nucleotide sequence capable of being hybridized to thesenucleotide sequences under stringent conditions, wherein the mutatednucleotide sequence encodes a protein having an endonuclease activitycapable of cleaving DNA in a pH range of from ca. 4.0 to ca. 7.6,independently from divalent cations.

The DNA encoding DLAD of the present invention is preferably one furthercontaining a nucleotide sequence encoding a signal peptide sequence atthe 5′ terminus of the nucleotide sequence as mentioned above. Morepreferably, the DNA encoding DLAD of the present invention consists ofthe nucleotide sequence of the nucleotide Nos. 213 to 1274 of thenucleotide sequence shown in Sequence Listing, SEQ ID NO: 2 or thenucleotide sequence of the nucleotide Nos. 1 to 1083 of the nucleotidesequence shown in Sequence Listing, SEQ ID NO: 4, or a nucleotidesequence capable of being hybridized to these nucleotide sequence understringent conditions, wherein the mutated nucleotide sequence encodes aprimary translation product of a protein having an endonuclease activitycapable of cleaving DNA in a pH range of from ca. 4.0 to ca. 7.6,independently from divalent cations.

In the context of the present invention, the “stringent conditions”means those under which a DNA having not less than about 60% homology tothe nucleotide sequence can be hybridized. The stringency can becontrolled by appropriately varying salt concentrations and temperaturesof the hybridization reaction and washing.

The DNA encoding DLAD of the present invention can be a DNA chemicallysynthesized based on the nucleotide sequence of the nucleotide Nos. 279to 1274 or the nucleotide Nos. 213 to 1274 of the nucleotide sequenceshown in Sequence Listing, SEQ ID NO: 2, or the nucleotide sequence ofthe nucleotide Nos. 82 to 1083 or the nucleotide Nos. 1 to 1083 of thenucleotide sequence shown in Sequence Listing, SEQ ID NO: 4.

The present invention also provides a recombinant vector containing aDNA encoding DLAD of the invention. The inventive recombinant vector isnot particularly limited as long as it can be maintained by replicationor autonomously replicated within various host cells, such asprokaryotic cells and/or eukaryotic cells, and encompasses plasmidvectors, viral vectors and the like. The recombinant vectors can beeasily prepared by ligating the above-mentioned DNA encoding DLAD toknown cloning vectors or expression vectors available in this technicalfield, using suitable restriction enzymes and a ligase, and further,linkers or adaptors as necessary. Examples of such vectors includepBR322, pBR325, pUC18, pUC19 etc. as a plasmid derived from Escherichiacoli; pSH19, pSH15 etc. as a plasmid derived from yeast; and pUB110,pTP5, pC194 etc. as a plasmid derived from Bacillus subtilis. Examplesof the viruses include bacteriophages such as λ phage, and animal andinsect viruses such as parvovirus (SV40, bovine papilloma virus (BPV)etc.), retrovirus (Moloney murine leukemia virus (MoMuLV) etc.),adenovirus (AdV), adeno-associated virus (AAV), vacciniavirus,vaculovirus, and the like.

Particularly, the present invention provides a DLAD expression vector inwhich a DNA encoding DLAD is placed under the control of a promoterfunctional in a desired host cell. The vector to be used is notparticularly limited as long as it contains a promoter region, which iscapable of functioning in various host cells such as prokaryotic and/oreukaryotic cells and regulating the transcription of a gene located atits downstream (e.g., when the host is Escherichia coli, trp promoter,lac promoter, leca promoter, etc., when the host is Bacillus subtilis,SPO1 promoter, SPO2 promoter, penP promoter, etc., when the host isyeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc.,and when the host is mammalian cell, viral promoters such as SV40 earlypromoter, MoMuLV long terminal repeat, adenovirus early promoter, etc.),and a termination signal of the transcription of said gene, i.e.,terminator region, wherein the promoter region and the terminator regionare ligated via a sequence containing at least one restriction enzymerecognition site, preferably unique restriction site that cleaves thevector only at this site. However, it is preferable that it furthercontain a selectable marker gene for selecting transformants (e.g., agene imparting resistance to a drug such as tetracycline, ampicillin,kanamycin, hygromycin and phosphinothricin, a gene complementingauxotrophic mutation etc.). Moreover, when the DNA encoding DLAD to beinserted does not contain an initiation codon or a termination codon, avector, which contains an initiation codon (ATG or GTG) and atermination codon (TAG, TGA or TAA) at the downstream of the promoterand the upstream of the terminator, respectively, is preferably used.

When bacteria is used as a host cell, in general, the expression vectorneeds to contain a replicable unit which allows autonomous replicationin the host cell, in addition to the above-mentioned promoter region andterminator region. The promoter region also contains an operator andShine-Dalgarno (SD) sequence near the promoter.

When a yeast, animal cell or insect cell is used as a host cell, theexpression vector preferably further contains enhancer sequences,non-translated regions on the 5′-side and 3′-side of DLAD mRNA, apolyadenylation site, and the like.

When DLAD is secreted into a culture medium of the transformant orproper glycosylation of the mature DLAD protein is desired but DNAencoding DLAD to be inserted does not have a sequence encoding signalpeptide, a secretory expression vector, further containing a suitablesignal codon following an initiation codon, is preferably used as avector.

When the DNA encoding DLAD of the present invention is isolated from agenomic DNA and obtained together with its native promoter andterminator regions, the expression vector of the present invention canbe prepared by inserting the DNA into a suitable site of a known cloningvector which can be maintained by replication or which can beautonomously replicated in a desired host cell. Since DLAD is expressedin a liver-specific manner in a preferable embodiment of the presentinvention, the expression vector constructed as mentioned above can bepreferably employed when a tissue- or organ-specific expression of DLADis desired (e.g., in the treatment of a CF patient with hepatic ductocclusion).

The present invention also provides a transformant obtained bytransforming a host cell with the above-mentioned DLAD expressionvector.

The host cell to be used in the invention is not particularly limited aslong as it is capable of adapting to the above-mentioned expressionvector and can be transformed therewith, and is exemplified by variouscells such as naturally occurring cells or artificially establishedmutant or recombinant cells conventionally used in the technical fieldof the present invention [e.g., bacteria (Escherichia coli, Bacillussubtilis, lactobacillus etc.), yeast (Saccharomyces, Pichia,Kluyveromyces etc.), animal cell and insect cell]. In view of the use ofDLAD as a medicament to be mentioned below, the host cells arepreferably mammalian cells, particularly the cells derived from human,monkey, mouse, rat, hamster etc., especially human-derived cells. To bespecific, exemplified are mouse-derived cells (COP, L, C127, Sp2/0,NS-1, NIH3 and T3), rat-derived cells, hamster-derived cells (BHK andCHO), monkey-derived cells (COS1, COS3, COS7, CV1 and Vero) andhuman-derived cells (HeLa, diploid fibroblast-derived cells, myelomacells and Namalwa).

The expression vector can be introduced into a host cell using a methodconventionally known. For example, the method of Cohen et al. [Proc.Natl. Acad. Sci. USA., 69, 2110 (1972)], protoplast method [Mol. Gen.Genet., 168, 111 (1979)] and competent method [J. Mol. Biol., 56, 209(1971)] can be used for bacteria; the method of Hinnen et al. [Proc.Natl. Acad. Sci. USA., 75, 1927 (1978)] or Lithium method [J.Bacteriol., 153, 163 (1983)] can be used for yeast; the method of Graham[Virology, 52, 456 (1973)] can be used for animal cell; and the methodof Summers et al. [Mol. Cell. Biol., 3, 2156–2165 (1983)] can be usedfor insect cell, for transformation.

The DLAD of the present invention can be produced by culturing atransformant containing the DLAD expression vector prepared as mentionedabove in a medium, and recovering DLAD from the resulting culture.

The medium to be used preferably contains carbon source and inorganic ororganic nitrogen source necessary for the growth of host cell(transformant). Examples of the carbon source include glucose, dextran,soluble starch and sucrose; examples of the inorganic or organicnitrogen source include ammonium salts, nitrates, amino acids, cornsteep liquor, peptone, casein, meat extract, soybean lees, potatoextract solution and the like. Where desired, other nutrient sourcessuch as inorganic salts (e.g., calcium chloride, sodiumdihydrogenphosphate and magnesium chloride), vitamins and antibiotics(e.g., tetracycline, neomycin, ampicillin and kanamycin) may be added.

Culture is performed by a method known in this field. Specific examplesof the medium and culture conditions to be used depending on the hostcell are shown in the following, which should not be construed aslimiting the culture conditions of the invention.

When the host is bacteria, actinomyces, yeast or fungus, a liquid mediumcontaining the aforesaid nutrient sources is suitable, with preferencegiven to a medium having a pH of 5 to 8. When the host is Escherichiacoli, preferable medium includes LB medium and M9 medium [Miller. J.,Exp. Mol. Genet, p.431, Cold Spring Harbor Laboratory, New York (1972)].In this case, culture can be typically performed at 14° C. to 43° C. forabout 3 to 24 hr with aeration and agitation as necessary. When the hostis Bacillus subtilis, culture can be typically performed at 30° C. to40° C. for about 16 to 96 hr with aeration and agitation as necessary.When the host is yeast, examples of the medium include Burkholderminimum medium [Bostian. K. L. et al., Proc. Natl. Acad. Sci. USA, 77,4505 (1980)], and pH is preferably 5 to 8. Culture can be typicallyperformed at 20° C. to 35° C. for about 14 to 144 hr with aeration andagitation as necessary.

When the host is animal cell, examples of the medium include minimumessential medium (MEM) containing about 5 to 20% fetal calf serum[Science, 122, 501 (1952)], Dulbecco's modified minimum essential medium(DMEM) [Virology, 8, 396 (1959)], RPMI1640 medium [J. Am. Med. Assoc.,199, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)]and the like. The pH of the medium is preferably about 6 to 8. Cultureis typically performed at 30° C. to 40° C. for about 15 to 72 hr withaeration and agitation as necessary.

When the host is an insect cell, examples of the medium include Grace'smedium containing fetal calf serum [Proc. Natl. Acad. Sci. USA, 82, 8404(1985)], and pH is preferably about 5 to 8. Culture is typicallyperformed at 20° C. to 40° C. for about 15 to 100 hr with aeration andagitation as necessary.

The DLAD can be purified by an appropriate combination of variousseparation techniques conventionally used, according to the fractionshaving DLAD activity. In a preferable embodiment of the invention, DLADis present both in cytoplasm and extracellularly (i.e., in medium).

The DLAD present in the medium in the culture can be obtained bycentrifuging or filtering the culture to give a culture supernatant(filtrate) and applying the culture supernatant to known separationmethods (e.g., salting out, solvent precipitation, dialysis,ultrafiltration, gel filtration, non-denatured PAGE, SDS-PAGE, ionexchange chromatography, hydroxylapatite chromatography, affinitychromatography, reversed-phase high performance liquid chromatographyand isoelectric focusing), as appropriately selected.

The DLAD present in the cytoplasm can be isolated and purified bycentrifuging or filtering the culture to harvest cells, suspending thecells in a suitable buffer, disrupting (lysing) the cells and organellemembranes by, for example, ultrasonication, lysozyme treatment,freeze-thawing, osmotic shock and/or treatment with surfactant such asTriton X-100, removing the debris by centrifugation or filtration togive a soluble fraction, and treating the soluble fraction according tothe methods mentioned above.

As a means for obtaining the recombinant DLAD rapidly and easily,preferably exemplified is a method which comprises adding a DNA sequenceencoding an amino acid sequence capable of adsorbing to a metal ionchelate (e.g., a sequence consisting of basic amino acids such ashistidine, arginine or lysine, preferably histidine) to a certain region(preferably C terminus) of the DLAD coding sequence by genemanipulation, allowing expression within a host cell, and recoveringDLAD from the DLAD active fraction in the cell culture by separationutilizing its affinity for a carrier immobilizing said metal ionchelate. The DNA sequence encoding an amino acid sequence capable ofadsorbing to a metal ion chelate can be introduced into the DLAD codingsequence by, for example, performing PCR amplification using a hybridprimer comprising said DNA sequence linked to the nucleotide sequenceencoding the C terminal amino acid sequence of DLAD, in the process ofcloning DNA encoding DLAD, or by inserting the DNA encoding DLAD inframe into an expression vector containing said DNA sequence before thetermination codon. The metal ion chelate adsorbent to be used forpurification is prepared by bringing a solution containing a transitionmetal (e.g., divalent ion of cobalt, copper, nickel or iron, ortrivalent ion of iron or aluminum, preferably divalent ion of cobalt ornickel) into contact with a ligand (e.g., a matrix onto whichiminodiacetate (IDA) group, nitrilotriacetate (NTA) group ortris(carboxymethyl)ethylenediamine (TED) group is attached) to allowbinding thereof with the ligand. The matrix part of the chelateadsorbent is not particularly limited as long as it is a conventionalinsoluble carrier.

The present invention provides a pharmaceutical composition containingthe inventive DLAD, DLAD expression vector or transformant expressingDLAD as an active ingredient, specifically an agent for the treatment ofchronic obstructive diseases (in particular cystic fibrosis) caused bythe accumulation of high concentration DNA and an agent for theprophylaxis and treatment of infectious diseases caused by viruses andthe like.

DNase I conventionally used for treating CF requires divalent cationsfor the expression of activity. However, it is speculated that theconcentration of divalent cations in lung cysts is not sufficiently highto allow expression of activation of DNase I. It is also considered thatthe pH in the inflammatory lesions in lung cysts inclines from neutralto acidic, though the optimal pH of DNase I is about 7.1. Furthermore,due to the fatal property that DNase is inhibited by G-actin present inlarge amounts in the sputum of CF patients, DNase I is almostineffective as an agent for treating CF. In contrast, the DLAD of thepresent invention is capable of cleaving DNA under acidic conditionsindependently from divalent cations. Furthermore, the DLAD of theinvention has excellent properties for exhibiting a high DNase activityin the inflammatory lesions in lung cysts of CF patients, in that it isnot inhibited by G-actin, it does not require any cofactor for theexpression of its activity, and it can exhibit its activity even in theneutral pH range.

DLAD has a high homology to FP-CEL1 [J. Virol., 72: 6742–6751 (1998)],which is a DNase II-related protein derived from fowlpox virus (FWPV).It is considered that, when a virus enters a cell infected with FWPV,the FWPV-derived DLAD homolog cleaves a DNA of the virus, thereby toexclude the competitive virus [J. Virol. (1998), supra]. Therefore, DLADcan also enhance the defensive function of a body against infectionswith virus etc., and is effective for the prophylaxis and treatment ofinfectious diseases. The infectious diseases that can be prevented ortreated are not particularly limited, and exemplified by those caused byhepatitis A, B and C viruses, human immunodeficiency virus, influenzavirus and herpes virus.

The administration subject of the inventive pharmaceutical compositionis not particularly limited as long as it is an animal in need of thetreatment of a chronic obstructive disease caused by the accumulation ofhigh concentration of DNA, or the prophylaxis and treatment of aninfectious disease caused by a virus or the like. It is preferably amammal, more preferably a mammal such as human, monkey, bovine, horse orporcine, especially human.

The pharmaceutical composition of the present invention containing aDLAD protein as an active ingredient can be formulated by admixing DLADwith a pharmaceutically acceptable carrier to give a liquid preparation,powder, granule, tablet, capsule, syrup, injection, aerosol or the like,and can be administered orally or parenterally.

The pharmaceutically acceptable carrier may include, but not limited to,excipients (e.g., sucrose, starch, mannitol, sorbit, lactose, glucose,cellulose, talc, calcium phosphate, calcium carbonate etc.), bindingagents (e.g., cellulose, methyl cellulose, hydroxypropylcellulose,polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol,sucrose, starch etc.), disintegrants (e.g., starch,carboxymethylcellulose, hydroxypropyl-starch, sodium glycol-starch,sodium bicarbonate, calcium phosphate, calcium citrate etc.), lubricants(e.g. magnesium stearate, aerosil, talc, sodium laurylsulfate etc.),flavors (e.g., citric acid, mentol, ammonium salt of glycyrrhizin,glycine, orange powders etc.), preservatives (e.g., sodium benzoate,sodium bisulfite, methylparaben, propylparaben etc.), stabilizers (e.g.,citric acid, sodium citrate, acetic acid, etc.), suspending agents(e.g., methyl cellulose, polyvinylpyrrolidone, aluminum stearate etc.),dispersing agents (e.g., surfactant etc.), diluents (e.g., water,physiological saline, orange juice etc.) and base waxes (e.g., cacaobutter, polyethylene glycol, white kerosine etc.).

Preferably, the pharmaceutical composition containing DLAD protein as anactive ingredient is a preparation for oral preparation, an injection oran aerosol preparation.

Preparations suitable for oral administration are liquid obtained bydissolving an effective amount of DLAD in diluents such as water,physiological saline and orange juice, capsule, sachet or tabletcontaining an effective amount of DLAD as solid or granule, suspensioncontaining an effective amount of DLAD suspended in an appropriatedispersion medium, and emulsion prepared by suspending a solutioncontaining an effective amount of DLAD dissolved in an appropriatedispersion medium and emulsifying the suspension.

The aerosol preparation may include one in which DLAD is compressed withdichlorodifluoromethane, propane or nitrogen and a non-compressedpreparation such as nebulizer and atomizer, and can be administered byinhalation or spraying into airways and the like.

Preparations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injectable liquids, which can containantioxidant, buffer, bacteriostat and isotonicity agent and the like,and aqueous and non-aqueous sterile suspensions, which can containsuspending agent, solubilizer, thickener, stabilizer, preservative andthe like. The DLAD preparations can be sealed in unit-dose or multi-dosecontainers such as ampoules or vials. It is also possible to lyophilize(freeze-dry) DLAD with a pharmaceutically acceptable carrier andpreserved in the form that requires dissolving or suspending in anappropriate sterile vehicle immediately prior to use.

The dose of the pharmaceutical composition containing the DLAD proteinof the invention varies depending upon the kind of disease to beprevented or treated, the progress of the disease, and the animalspecies, drug-tolerance, weight and age of the administration subject,which is typically 1 to 10,000 I.U./kg body weight, preferably 10 to1,000 I.U./kg body weight, daily for an adult, which can be administeredin a single dose or several doses.

The present invention also provides a pharmaceutical compositioncontaining the DLAD expression vector of the present invention as anactive ingredient. Since the treatment of CF using DNases is notfundamental but suppressive, continuous supply of DLAD to theinflammatory lesions in the lung cysts is required. Accordingly, a genetherapy, in which the DLAD expression vector is introduced into cells ator around the inflammatory lesion, is effective as a sustainabletherapeutic method for CF. For the purpose of preventing viral infectionof livestock and the like, a transgenic animal having enhancedpreventive function against infection, can be produced by introducingthe DLAD expression vector into the embryonic cells.

The vector to be used can be selected according to the administrationsubject, and examples of vectors preferably administered to humaninclude viral vectors such as retrovirus, adenovirus andadeno-associated virus. Adenovirus is particularly preferable as a DLADgene transfer vector for the treatment of CF, because it has a very highgene transfer efficiency, can be introduced even into non-dividingcells, and is trophic for the respiratory epithelium. However, since theintegration of the introduced gene into the host chromosome is extremelyrare, the gene expression is transient and typically lasts for about 4weeks. In view of the sustainability of the therapeutic effect, the useof an adeno-associated virus is also preferable, which has a relativelyhigh gene transfer efficiency, can be introduced even into non-dividingcells and can be integrated into chromosome via inverted terminalrepeats (ITRs).

Examples of pharmaceutically acceptable carriers contained in thepharmaceutical composition containing a DLAD expression vector as anactive ingredient may be those for the above-mentioned pharmaceuticalcomposition containing the DLAD protein.

The vector can be introduced by either an ex vivo method, whichcomprises isolating the target cells from the administration subject,culturing, transferring the vector thereto and implanting the cells backinto the subject, or an in vivo method, which comprises directlytransferring the vector into the body of the administration subject.When the in vivo method is used, the administration of the vector viaintravenous injection or the like may raise a problem of theantigenicity of the viral vector, but the undesired effects caused bythe presence of the antibody can be reduced by topically injecting thevector into the organ/tissue containing the target cells (in situmethod).

When a non-viral vector is used as a vector, the DLAD expression vectorcan be introduced using macromolecule carriers such as liposome andpolylysine-DNA-protein conjugate.

The present invention also provides a pharmaceutical composition, whichcomprises a host cell containing the DLAD expression vector of theinvention as an active ingredient. The host cells to be used may includeautogenous cells, which are isolated as target cells from theadministration subject in the ex vivo method of the gene therapy usingthe above-mentioned DLAD expression vector, cells isolated from thesyngeneic or allogeneic individuals, or established cell lines derivedfrom these cells by subculture. In another embodiment, a transformantobtained by transforming a host cell, which is normally present in thenasal cavity, pharynx, oral cavity, intestinal tract, skin, vagina andthe like of the administration target animal, with a DLAD expressionvector according to a conventional method, can be delivered to the sitewhere the host cell is normally present in the administration subject.

The dose of the pharmaceutical composition containing the DLADexpression vector or the host cell, which expresses this vector of thepresent invention, as an active ingredient, is preferably one capable ofexpressing DLAD in the body of an animal to which it is administered,the dose corresponding to an amount suitable for allowing the expressionto be achieved when the DLAD protein itself is administered.

The present invention is further explained in detail by way of Examplesin the following. These are mere examples, which in no way limit thescope of the present invention.

EXAMPLE 1 Cloning and Sequence Analysis of Mouse DLAD cDNA

The EST subdivision of the NCBI GenBank database was screened for ESTencoding an amino acid sequence homologous to the deduced amino acidsequence of human DNase II (GenBank AF060222) using the tblastn program.As a result, a mouse EST clone (GenBank AI048641) was identified. Basedon the sequence of the EST clone, the following two oligonucleotideprimers (GSP2/mD and GSP1/mD) were synthesized. Furthermore, thefollowing oligonucleotide (AP1) was synthesized as a linker primer.

GSP2/mD:

-   5′-AATGAATATGGTGAAGCTGTGGACTGG-3′ (Sequence Listing, SEQ ID NO: 5)    (sequence identical to the nucleotide sequence of the nucleotide    Nos. 300 to 326 of the nucleotide sequence shown in Sequence    Listing, SEQ ID NO: 2)    GSP1/mD:-   5′-CCATCGTTGTATATTAGATAGGCTGTG-3′ (Sequence Listing, SEQ ID NO: 6)    (sequence complementary to the nucleotide sequence of the nucleotide    Nos. 509 to 535 of the nucleotide sequence shown in Sequence    Listing, SEQ ID NO: 2)    AP1:-   5′-CCATCCTAATACGACTCACTATAGGGC-3′ (Sequence Listing, SEQ ID NO: 7)

Using the following primers containing oligo dT:

-   5′-TTCTAGAATTCAGCGGCCGC(T₃₀)VN-3′ (Sequence Listing, SEQ ID NO: 8)    (wherein V is G, A or C, and N is G, A, C or T)    a reverse transcription reaction was performed using C57black/6    mouse liver-derived poly A(+) RNA as a template to generate a single    strand CDNA (antisense strand). The sense strand was further    synthesized according to a conventional method to give a double    strand DNA followed by ligation of the linker DNA containing the AP1    sequence to its both ends. Using this cDNA as a template, 3′ RACE    reaction was performed with GSP2/mD as a sense primer and AP1 as an    antisense primer, and then 5′ RACE reaction was performed with AP1    as a sense primer and GSP1/mD as an antisense primer. Marathon cDNA    Amplification kit (Clontech) was used in these RACE reactions. Each    amplification product was subcloned into pBluescript KS+    (Stratagene), and the nucleotide sequence of each insert was    determined by cycle sequencing using 7-Deaza Thermo Sequenase kit    (Amasham) and DSQ1000L DNA sequencer (Shimazu).

As a result, it was revealed that the mRNA containing the EST clonesequence with homology to DNase II as a partial sequence consists of thenucleotide sequence shown in Sequence Listing, SEQ ID NO: 2. The 3′ RACEreaction resulted in two cDNA fragments that differ in theirpolyadenylation sites. Poly A-added signal consensus sequences (AATAAA)are found at 14 nucleotides upstream of the first polyadenylation site(the nucleotide No. 1409) and 18 nucleotides upstream of the secondpolyadenylation site (the nucleotide No. 1634), which are consistentwith this observation.

Sequence analysis revealed that this cDNA sequence contains an ORF of1065 bp (the nucleotide Nos. 213 to 1277) encoding 354 amino acids of anovel polypeptide. The ORF had 37.1% amino acid identity with DNase II.The molecular weight of the polypeptide encoded by the ORF, which iscalculated from the deduced amino acid sequence, was 40,767. On thebasis of the enzymological properties (see below) of the recombinantlyproduced protein, the present inventors designated this novel protein asDNase II-Like Acid DNase (DLAD).

DLAD is a highly basic protein (isoelectric point: 9.67) containing 8potential N-glycosylation sites [the Asn residue of Asn-Xaa-Thr/Ser (Xaais an optional amino acid); the amino acid Nos. 48, 55, 76, 92, 107,186, 249 and 297 of the amino acid sequence shown in Sequence Listing,SEQ ID NO: 1]. The possible N terminal signal peptide was predicted tobe the first 22 amino acids (the amino acid Nos. −22 to −1) by vonHeijne's method [Nucleic Acids Res., 14: 4683–4690 (1986)].

A homology search of the GenBank database revealed that DLAD has 32.1%,25.1% and 19.4% amino acid identities with three proteins, C07B5.5,F09G8.2 and K04H4.6, encoded by putative ORFs of a nematode C. elegansgenome, respectively. Furthermore, DLAD shares 37.5% amino acid identitywith FP-CEL1 encoded by the third ORF of FWPV genome, which value ishigher than the identity between DNase II and FP-CEL1 (28.4%).

EXAMPLE 2 Tissue Distribution of DLAD mRNA

Northern blot analysis was performed to assess the expression of DLADmRNA in various mouse tissues. Total RNA was extracted from each tissue(brain, thymus, lung, heart, liver, stomach, small intestine, spleen,kidney and testis) of adult mouse with TRIzol reagent (Gibco BRL). EachRNA aliquot (15 μg) was subjected to 1% agarose-formamide gelelectrophoresis and blotted onto a Biodyne-A membrane (Paul). Thismembrane was subjected to the hybridization with a ³²P-labeled probe,obtained by random priming of Xho I digestion fragment of pcDLAD-Myc-His(see below), which is a vector carrying the DNA encoding a DLAD-Mycfusion protein with a histidine tag, in hybridization solutionconsisting of 5×SSPE, 5×Denhardt's solution, 50% formamide, 0.1% SDS and100 μg/ml heat-denatured salmon sperm DNA at 42° C. overnight. Thehybridization solution was removed after the reaction, and the membranewas washed with 0.1×SSC, 0.1% SDS at 50° C. and exposed to X-ray film at−80° C. for 5 days using an intensifying screen. As a result, a singleband corresponding to the 1.9 kb DLAD mRNA was detected only in theliver. This liver-specific expression is contrastive with the poororgan-specificity of DNase II, clearly suggesting the distinctphysiological function of DLAD from that of DNase II.

EXAMPLE 3 Localization of DLAD Protein

The primary structure analysis of DLAD in Example 1 revealed that theprimary translation product of DLAD has a hydrophobic domain satisfyingthe requirement for a signal peptide at the N terminus. That is, it wassuggested that DLAD was a secretory protein. Then, to confirm this, aDLAD expression vector was introduced into a human cultured cellfollowed by comparison of the presence ratio ofintracellular/extracellular DLADS. The subcellular localization of DLADpresent within the cell was also analyzed.

(1) Construction of DLAD Expression Vector

RT-PCR reaction was performed with C57black/6 mouse liver-derived polyA(+) RNA as a template, using the following pair of primers to generatea cDNA fragment containing DLAD ORF without termination codon, and theresulting cDNA fragment was subcloned into pBluescript KS+.

Senseprimer:

-   5′-CTCGAGCCACCATGACAGCAAAGCCTCTAAGAACA-3′ (Sequence Listing, SEQ ID    NO: 9)    (sequence with a linker sequence containing Xho I recognition site    (CTCGAG) added to the 5′ terminus of the nucleotide sequence of the    nucleotide Nos. 213 to 236 of the nucleotide sequence shown in    Sequence Listing, SEQ ID NO: 2)    Antisense Primer:-   5′-CTCGAGACTTACAGAACCCATAACGGAGAT-3′ (Sequence Listing, SEQ ID NO:    10)    (sequence with a linker sequence containing Xho I recognition site    (CTCGAG) added to the 5′ terminus of the sequence complementary to    the nucleotide sequence of the nucleotide Nos. 1252 to 1274 of the    nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2)

After confirming the sequence by cycle sequencing, the insert DNA wasexcised by Xho I digestion and re-cloned into the Xho I sites ofpcDNA3-Myc-His C (Invitrogen) and pEGFP-N3 (Clontech) to generateexpression vectors encoding DLAD with C terminal Myc and histidine tags(pcDLAD-Myc-His) and encoding DLAD-GFP (Green Fluorescence Protein)fusion protein (pDLAD-GFP), respectively. For comparison, an expressionvector encoding DNase II with C terminal Myc and histidine tags(pcDNaseII-Myc-His) was generated by the same procedure.

Furthermore, to assess the secretion efficiency of DLAD signal peptide,an expression vector encoding a chimeric protein, in which the signalpeptide of DLAD is replaced with that of DNase II (pcDNaseII/DLAD), wasproduced in the following procedure. DNA fragments encoding the DNase IIsignal peptide and encoding DLAD without signal peptide were amplifiedby PCR respectively using the following two pairs of primers. Theresulting fragments were ligated after Hae II digestion and subclonedinto pBluescript KS+. After confirming the sequence, the insert wasre-cloned into pcDNA3-Myc-His C to give pcDNaseII/DLAD.

Amplification of DNase II Signal Peptide Coding Sequence

Sense Primer:

-   5′-CTCGAGCCACCATGATCCCGCTGCTGCTGGCA-3′ (Sequence Listing, SEQ ID NO:    11)    Antisense Primer:-   5′-GCAGGTCAGGGCGCCGGC-3′ (Sequence Listing, SEQ ID NO: 12)    Amplification of Signal Peptide(−) DLAD Coding Sequence    Sense Primer:-   5′-AGCTAGGCGCCCTCTCATGCAGAAATGAA-3′ (Sequence Listing, SEQ ID NO:    13)    Antisense Primer:-   5′-CTCGAGACTTACAGAACCCATAACGGAGAT-3′ (Sequence Listing, SEQ ID NO:    10)    (2) Transfection and Western Blot Analysis

HeLa S3 cells (2×10⁵) grown in RPMI 1640 medium supplemented with 10%fetal calf serum were transfected individually with 1 μg ofpcDLAD-Myc-His, pcDNaseII/DLAD or pcDNaseII-Myc-His using FuGene6transfection reagent (Boehringer). After incubating for 48 hr, theculture supernatant (extracellular fraction) and the cell were collectedseparately. The cells were homogenized in 2 ml of ice-cold buffer A [100mM Tris-HCl (pH7.8), 3 mM MgCl₂, 1 mM 2-mercaptoethanol and 0.3 mM PMSF]containing 0.1% Nonidet P-40 with a Teflon-glass homogenizer by 10strokes. The homogenate was centrifuged at 10,000×g for 10 min and thesupernatant was recovered as intracellular (cytoplasmic) fraction.Recombinant proteins with histidine-tags were purified from theintracellular and extracellular fractions, respectively, using Ni-NTAspin column (Quiagen) according to the manufacture's protocol. Afterconcentration with Ultrafree MC (Millipore), aliquots of the eluateswere subjected to 10% SDS-PAGE and transferred onto Immobilon P membrane(Millipore). The membrane was blocked in TBST [20 mM Tris-HCl (pH8.0),400 mM NaCl and 0.05% (w/v) Triton X-100] containing 2.5% bovine serumalbumin for 1 hr and reacted with mouse anti-Myc antibody (Novagen).After washing with TBST, the antibody retained on the membrane wasdetected using anti-mouse IgG (Promega) labeled with an alkalinephosphatase. The staining image was scanned with a CCD camera (Atto) andthe optical densities of the bands were quantified by densitometry (NIHimage 1.60). The results are shown in FIG. 1.

The DLAD with Myc and His-tags was detected as a single band of 58 kDa.Calculating the molecular weight (about 3 kDa) of the Myc and His-tagsadded to C terminus, the molecular weight of DLAD per se is estimated asabout 55 kDa by SDS-PAGE, which value is larger than the molecularweight 38,452 Da deduced from the amino acid sequence (the amino acidNos. 1 to 332) of DLAD. This appears to be due to glycosylation.

While about 80% of DNase II was secreted extracellularly, about 70% ofDLAD retained within the cells and about 30% was found in theextracellular fraction (FIG. 1). Since DLAD is as stable as DNase II inthe medium for HeLa S3 cells, the low presence ratio of extracellularDLAD is not due to the rapid degradation of DLAD in the medium. Theinefficient secretion of DLAD was not improved by replacing its signalpeptide with that of DNase II, which indicates that this inefficiency isnot due to weak secretion signal of DLAD itself. Thus, it is suggestedthat some targeting motif(s) as intracellular retention signal(s) existin the mature DLAD protein.

(3) Fluorescence Microscopic Analysis of DLAD-GFP Fusion Protein

HeLa S3 cells (2×10⁵) grown on a coverslip were transfected withpDLAD-GFP (1 μg) in the same manner as in (2) above. The cells wereincubated for 48 hr and fixed with 1% glutaraldehyde in PBS(−) at roomtemperature for 10 min. After washing the coverslip with PBS(−), thecell nuclei were stained with 1 mM Hoechst 33258 in PBS(−) and theimages of GFP and DNA were observed by a fluorescence microscope(Olympus). As a control, HeLa S3 cells engineered to express GFP alonewas observed in the same manner. As a result, while GFP gave a diffuseimage expanded both in the cytoplasmic and nuclear regions in cytoplasm,DLAD-GFP fusion protein was detected as a granular pattern. Thesesuggest that the intracellular DLAD is localized in cytoplasm andtargeted some organelle. A motif search using PSORT II program revealedthat DLAD contains no transition signals for mitochondria or nuclei.Thus, it is speculated that a possible target organelle for thecytoplasmic DLAD is acidic organelle such as lysosome or peroxysome.

EXAMPLE 4 Analysis of the Enzymological Properties of DLAD

(1) Purification of Recombinant DLAD

In the same manner as in (2) of Example 3, HeLa S3 cells (5×10⁶) weretransfected with 25 μg of pcmDLAD-Myc-His or pcmDNaseII-Myc-His,individually. These plasmids encode the mature proteins of DLAD andDNase II, respectively, wherein Myc and His-tags are added to their Ctermini. After incubation of the cells for 48 hr, recombinant DLAD andrecombinant DNase II were purified by IMAC using Ni-NTA spin column, inthe same manner as in (2) of Example 3. The purified DLAD (or DNase II),which was eluted in 300 μl of elution buffer [50 mM sodium phosphate (pH8.0) containing 250 mM imidazole and 300 mM NaCl], was dialyzed against20 mM Mes-NaOH containing 1 mM 2-mercaptoethanol and used in thefollowing assay for enzyme activities. The Sample obtained by treatingHeLa S3 cells transfected with empty vector in the same manner was usedas a control.

In the following experiments, the assay for DNase activity was performedas below, unless otherwise described. 20 μl of reaction mixture [50 mMMes-NaOH (pH 5.2), 1 mM 2-mercaptoethanol, 1 unit of enzyme, 500 ngsupercoiled or EcoR I-digested linear pBluescript KS+] was prepared onice and incubated at 45° C. for 20 min. The reaction was terminated withphenol/chloroform and 5 μl aliquot of the reaction mixture was subjectedto 1% agarose gel electrophoresis. After ethidium bromide staining, theimage was scanned with CCD camera (Atto) under UV transillumination andoptical density of the band corresponding to the full length substrateDNA was quantified by densitometry (NIH image 1.60). DNase activity wasdetermined using reduction of the band intensity corresponding to thefull length substrate DNA as an index. In the present invention, 1 unitof DLAD and DNase II activities are defined as their amounts required todecrease the band intensity corresponding to 200 ng of the full lengthsubstrate DNA under the above-mentioned reaction conditions.

(2) Divalent Cation-dependency

Using a supercoiled plasmid as a substrate, DLAD activities in thepresence and absence of a divalent cation chelator were determined underacidic (50 mM Mes-NaOH, pH 5.2) and neutral (50 mM Mops-NaOH, pH 7.2)conditions, respectively. 1 mM of EDTA and EGTA were used individually,as chelators. As a result, DLAD exhibited an endonuclease activitycatalyzing the degradation of the supercoiled plasmid DNA under both pHconditions. However, DLAD activity under acidic condition was muchhigher than that under neutral condition. The addition of divalentcation chelator, EDTA or EGTA had no effect on DLAD activity regardlessof pH ranges. Thus, DLAD was demonstrated to be a divalentcation-independent acid DNase. No endonuclease activity was detected inthe same assays using the sample derived from HeLa S3 cells transfectedwith empty vector, indicating that the DLAD activity detected is not dueto contamination of the endogenous DNases in HeLa S3 cells.

(3) Active pH Range and Optimal pH

Using a supercoiled plasmid as a substrate, DLAD and DNase II activitieswere determined under the above-mentioned standard conditions exceptvarying kinds of buffers and pH [i.e., acetate-NaOH (pH 4.0 and 4.4),Mes-NaOH (pH 4.8, 5.2, 5.6, 6.0 and 6.4) and Mops-NaOH (pH 6.4, 6.8, 7.2and 7.6)]. As a result, DLAD showed its DNase activity in all of the pHranges examined with a maximum at pH 5.2 in Mes-NaOH. However, DNase IIactivity was observed only at pH 5.6 or below.

(4) Sensitivity Against Inhibitors

DLAD activities were determined in the presence of variousconcentrations of DNase inhibitors [MgCl₂ (FIG. 2A, ∘), MgSO₄ (FIG.2A,), aurintricarboxylic acid (FIG. 2B), G-actin (FIG. 2C), CoCl₂ (FIG.2D, ▪), NiCl₂ (FIG. 2D, Δ) and ZnCl₂ (FIG. 2D,)] under theabove-mentioned standard conditions to analyze its sensitivity againstthe inhibitors. As a result, high concentrations of MgCl₂ inhibited DLADactivity (IC₅₀=13 mM). MgSO₄, an inhibitor of DNase II, inhibited DLADmore efficiently than MgCl₂ (IC₅₀=7 mM), indicating that SO₄ ²⁻ ion iseffective to inhibit DLAD. Aurintricarboxylic acid, a general inhibitorof nucleases, strongly inhibited DLAD (IC₅₀=6 μM), whereas G-actin, aninhibitor of DNase I, did not inhibit DLAD. The comparison of thesensitivity to three divalent metal ions revealed that DLAD is mostsensitive to Zn²⁺ (IC₅₀=0.2 mM). In contrast, it is known that there arelittle differences between these three divalent metal ions in inhibitoryeffect on DNase II [J. Biochem., 87: 1097–1103 (1980)].

(5) Mode of DNA Hydrolysis

Using a supercoiled pBluescript KS+ as a substrate, enzyme reaction ofDLAD or DNase II was performed under the above-mentioned standardconditions. After terminating the reaction, contaminants were removed byphenol/chloroform treatment to isolate degraded plasmid DNA. The 3′ endswere labeled in 50 μl of a reaction solution consisting of 20 units ofterminal deoxynucleotidyl transferase (Toyobo), 0.83 mCi/ml [γ-³²P]dCTP,100 mM sodium cacodylate (pH 7.2), 0.2 mM DTT and 1 mM CoCl₂. The 5′ends were labeled in 50 μl of a reaction solution consisting of 20 unitsof polynucleotide kinase (Toyobo), 0.83 mCi/ml [γ-³²p]ATP, 50 mMTris-HCl (pH 8.0), 10 mM MgCl₂ and 10 mM 2-mercaptoethanol. Theend-labeling treatments were performed as to both DNAs with and withoutpretreatment with 20 units of calf intestinal alkaline phosphatase(Takara) in the presence of 50 mM Tris-HCl (pH 8.0) and 1 mM MgCl₂ toremove the phosphoryl groups in the ends of DNAs, respectively.Unincorporated nucleotides were removed by ethanol precipitation. Thelabeled DNA was subjected to 1% agarose gel electrophoresis, transferredonto nylon membrane and analyzed with BAS 1500 image analyzer (FujiFilm). As a result, in both DNA fragments treated with DLAD and DNaseII, the 5′ ends were labeled regardless of pretreatment with alkalinephosphatase, whereas the 3′ ends could be labeled only after removal ofthe phosphoryl groups. Thus, it is revealed that DLAD catalyzes DNAhydrolysis to generate 3′-P/5′-OH ends as DNaseII does.

EXAMPLE 5 Suppression Effects of DLAD on the Expression of Foreign DNA

(1) Isolation of Rat DLAD cDNA

Using the same strategy as in Example 1, a rat EST clone (GenBankAF178974) encoding the amino acid sequence with homology to the deducedamino acid sequence of human DNase II was identified. Oligonucleotideprimers were synthesized based on the nucleotide sequence of this clone,followed by the RACE with liver poly A(+) RNA derived from Wister rat,which is connately pigment-deficient (albino), as a template to clonethe full length rat DLAD cDNA. From the sequence analysis by aconventional method, it was deduced that this cDNA contains an ORFencoding 356 amino acids with a signal peptide consisting of 22 aminoacids at the N terminus. It was found to have 83.3% DNA identity and70.8% amino acid identity to mouse DLAD. An expression vector encodingrat DLAD with Myc and His-tags at the C terminus (prDLAD-Myc-His) wasconstructed by the same strategy as in (1) of Example 3. HeLa S3 cellswere transfected with this vector to generate a recombinant rat DLAD.Characterization of the obtained recombinant protein confirmed that thisprotein has properties similar to mouse DLAD in active pH range,divalent cation-dependency, mode of DNA cleavage, sensitivity toinhibitors and the like.

(2) Suppression of the Expression of Foreign Reporter Gene in DLADExpressing HeLa Cells

HeLa S3 cells (2×10⁵) grown on a coverslip were co-transfected withprDLAD-Myc-His (1 μg) and pcDNA3.1-Myc-His/lac Z (β-galactosidaseexpression vector; Invitrogen) or pEGFP-N3 (Clontech; supra) (0.5 μg),using the same method as in (2) of Example 3. Assay for GFP activity wasperformed according to the method described in (3) of Example 3.β-galactosidase activity was determined with β-galactosidase assaysystem (Promega) according to the attached protocol. The sameexperiments were performed using an expression vector encoding rat DNaseII in place of DLAD with Myc and His-tags at the C terminus(prDNaseII-myc-His). An empty vector, pcDNA3.1-Myc-His (Invitrogen), wasused as a control. The results are shown in FIG. 3.

As seen from the figure, while DNase II has no effect on the expressionof the foreign reporter genes, DLAD suppressed the expression of theseforeign genes by about 20 to 25% versus control. These observationssuggest that DLAD has an effect to act on heterologous DNA entering intocells such as viral DNA, and degrade and remove the DNA.

EXAMPLE 6 Isolation of Human DLAD cDNA

Using the same strategy as in Example 1, a human EST clone (GenBank No.AA988125) encoding the amino acid sequence with homology to the aminoacid sequence of mouse DLAD was identified. On the basis of thenucleotide sequence of this clone, the following primers:

GSP2/h2L:

-   5′-AACTGCTCCCTTCCTTACCATGTCTAC-3′ (Sequence Listing, SEQ ID NO: 14)    (sequence identical to the nucleotide sequence of the nucleotide    Nos. 832 to 858 of the nucleotide sequence shown in Sequence    Listing, SEQ ID NO: 4)    GSP1/h2L:-   5′-GAAGGCTTGGTGTGGACTCCGATTTAG-3′ (Sequence Listing, SEQ ID NO: 15)    (sequence complementary to the nucleotide sequence of the nucleotide    Nos. 973 to 999 of the nucleotide sequence shown in Sequence    Listing, SEQ ID NO: 4)    were synthesized and, further using the above-mentioned linker    primer AP1 together with these primers, RACE was performed with    human liver-derived poly A(+) RNA as a template to clone the full    length human DLAD cDNA. From the sequence analysis by a conventional    method, it was deduced that this cDNA contains an ORF (Sequence    Listing, SEQ ID NO: 4) encoding 361 amino acids (Sequence Listing,    SEQ ID NO: 3) with a signal peptide consisting of 27 amino acids at    the N terminus. It was found to have 75.1% DNA identity and 65.4%    amino acid identity to mouse DLAD. An expression vector encoding    human DLAD with Myc and His-tags at the C terminus (phDLAD-Myc-His)    was constructed by the same strategy as in (1) of Example 3. HeLa S3    cells were transfected with this vector to generate a recombinant    human DLAD. Characterization of the obtained recombinant protein    confirmed that this protein has properties similar to mouse DLAD in    active pH range, divalent cation-dependency, mode of DNA cleavage,    sensitivity to inhibitors and the like.    Free text in Sequence Listing-   SEQ ID NO: 5: Oligonucleotide designed to act as sense primer for    amplifying 3′-terminal of mouse DLAD cDNA.-   SEQ ID NO: 6: Oligonucleotide designed to act as antisense primer    for amplifying 5′-terminal of mouse DLAD cDNA.-   SEQ ID NO: 7: Oligonucleotide designed to act as linker primer for    amplifying 5′- and 3′-terminals of DLAD cDNA.-   SEQ ID NO: 8: Oligonucleotide designed to act as primer for reverse    transcription of mouse DLAD RNA, wherein v is g, a or c and n is g,    a, c or t.-   SEQ ID NO: 9: Oligonucleotide designed to act as sense primer for    amplifying full length mouse DLAD cDNA.-   SEQ ID NO: 10: Oligonucleotide designed to act as antisense primer    for amplifying full length mouse DLAD cDNA.-   SEQ ID NO: 11: Oligonucleotide designed to act as sense primer for    amplifying coding sequence of DNase II signal peptide.-   SEQ ID NO: 12: Oligonucleotide designed to act as antisense primer    for amplifying coding sequence of DNase II signal peptide.-   SEQ ID NO: 13: Oligonucleotide designed to act as sense primer for    amplifying coding sequence of DLAD lacking its signal peptide.-   SEQ ID NO: 14: Oligonucleotide designed to act as sense primer for    amplifying 3′-terminal of human DLAD cDNA.-   SEQ ID NO: 15: Oligonucleotide designed to act as antisense primer    for amplifying 5′-terminal of human DLAD cDNA.

This application is based on application No. 11-230870 filed in Japan,the contents of which are incorporated hereinto by reference.

All of the references cited herein containing patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication was specifically described herein.

1. An isolated DNA consisting of a nucleotide sequence of nucleotideNos. 279 to 1274 of SEQ ID NO:2.
 2. An isolated DNA consisting of anucleotide sequence of nucleotide Nos. 213 to 1274 of SEQ ID NO:2.
 3. Arecombinant vector comprising the DNA of: (i) a nucleotide sequence ofnucleotide Nos. 279 to 1274 of SEQ ID NO:2, or (ii) a nucleotidesequence of nucleotide Nos. 213 to 1274 of SEQ ID NO:2.
 4. An expressionvector comprising (a) the DNA of: (i) a nucleotide sequence ofnucleotide Nos. 279 to 1274 of SEQ ID NO:2, or (ii) a nucleotidesequence of nucleotide Nos. 213 to 1274 of SEQ ID NO:2, and (b) apromoter operably linked to said DNA.
 5. A transformant obtained bytransforming a host cell with the expression vector of claim
 4. 6. Amethod for producing the deoxyribonuclease, which comprises culturingthe transformant of claim 5 in a medium and recovering thedeoxyribonuclease from a resulting culture.
 7. An isolated DNA encodinga deoxyribonuclease comprising: an amino acid sequence of amino acidNos. 1 to 332 of SEQ ID NO:
 1. 8. An expression vector comprising theDNA of claim 7 and a promoter operably linked to said DNA.
 9. Atransformant obtained by transforming a host cell with the expressionvector of claim
 8. 10. A method for producing a deoxyribonuclease, whichcomprises culturing the transformant of claim 9 in a medium andrecovering the deoxyribonuclease from a resulting culture.
 11. Anisolated DNA consisting of a nucleotide sequence that hybridizes to thecomplement of the nucleotide sequence of nucleotides Nos. 279 to 1274 ofSEQ ID NO:2 under highly stringent conditions, which encodes adeoxyribonuclease having an endonuclease activity that cleaves DNA, at apH range of from ca. 4.0 to ca. 7.6.
 12. An isolated DNA consisting of anucleotide sequence that hybridizes to the complement of the nucleotidesequence of nucleotides Nos. 213 to 1274 of SEQ ID NO:2 under highlystringent conditions, which encodes a primary translation product of adeoxyribonuclease which in mature protein form has an endonucleaseactivity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6. 13.A recombinant vector comprising the DNA of: (i) a nucleotide sequencethat hybridizes to the, complement of the nucleotide sequence ofnucleotides Nos. 279 to 1274 of SEQ ID NO:2 under highly stringentconditions, which encodes a deoxyribonuclease having an endonucleaseactivity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6, or(ii) a nucleotide sequence that hybridizes to the complement of thenucleotide sequence of nucleotides Nos. 213 to 1274 of SEQ ID NO:2 underhighly stringent conditions, which encodes a primary translation productof a deoxyribonuclease which in mature protein form has an endonucleaseactivity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6. 14.An expression vector comprising (a) the DNA of: (i) a nucleotidesequence that hybridizes to the complement of the nucleotide sequence ofnucleotides Nos. 279 to 1274 of SEQ ID NO:2 under highly stringentconditions, which encodes a deoxyribonuclease having an endonucleaseactivity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6, or(ii) a nucleotide sequence that hybridizes to the complement of thenucleotide sequence of nucleotides Nos. 213 to 1274 of SEQ ID NO:2 underhighly stringent, conditions, which encodes a primary translationproduct of a deoxyribonuclease which in mature protein form has anendonuclease activity that cleaves DNA, at a pH range of from ca. 4.0 toca. 7.6, and (b) a promoter operably linked to said DNA.
 15. Atransformant obtained by transforming a host cell with the expressionvector of claim
 14. 16. A method for producing the deoxyribonuclease,which comprises culturing the transformant of claim 15 in a medium andrecovering the deoxyribonuclease from a resulting culture.
 17. Anisolated DNA comprising a nucleotide sequence of the nucleotide Nos. 279to 1274 of SEQ ID NO:
 2. 18. An isolated DNA comprising a nucleotidesequence of the nucleotide Nos. 213 to 1274 of SEQ ID NO: 2.